<b>Objectives</b><br/> The cellular function of cyclophilin A (CypA) differs between organisms, even though CypA is conserved in both prokaryotes and eukaryotes. The purpose of this study was to elucidate the role of activated CypA isoform CPR1 in the antioxidative mechanisms of <i>Saccharomyces cerevisiae</i> under menadione (MD)-induced oxidative stress.<br/><b>Methods</b><br/> Four <i>S. cerevisiae</i> strains, KNU5377Y (kwt) and BY4741 (bwt), and their isogenic <i>cpr1</i>⊿ mutant strains (kc1 and bc1), were treated with MD, at a concentration ranging between 0.25 and 0.4 mM. Cpr1-mediated antioxidative effects were analyzed by measuring the levels of cellular glutathione (GSH) and ascorbate (AsA)-like molecules in yeast.<br/><b>Results</b><br/> GSH and AsA-like reductant molecule concentrations were more reduced in the presence of MD in the kc1 strain than in the kwt strain; whereas, there was no significant difference between the bwt and bc1 strains under the same conditions. In kc1 strain samples, we observed a reduction in the expression of proteins related both to GSH synthesis and the recycling system, and simultaneously, downregulation of GSH synthetase and GSH reductase activities were also evident. Oxidative stress in the kc1 strain was alleviated by the application of the GSH and AsA analog.<br/><b>Conclusion</b><br/> These results indicate that activated Cpr1 modulates the response of antioxidant molecules involved in cellular redox homeostasis of KNU5377Y during oxidative stress induced by MD.